The human immunodeficiency virus (HIV) causes a lethal syndrome (acquired immunodeficiency syndrome) characterized by CD4+ T cell depletion and resultant immunodeficiency. HIV has caused a worldwide epidemic that has killed millions of people and continues to infect about 40,000 people each year in this country.
The long-term goal of our research program is to provide improved treatments for people with HIV/AIDS. Existing therapies are highly effective at rendering viral particles non-infectious and thus reducing viral loads. However, none of the current drugs are capable of destroying infected cells. Many infected cells have short half-lives and die within days or months. Others, however, remain in a state that is resistant to the immune system and can persist for years. To provide better therapies, our focus has been on understanding the molecular mechanisms of viral persistence within cellular reservoirs. The development of drugs that will inhibit these pathways will bring us closer to a cure.
To this end, our research program focuses on major viral mechanisms of resistance to the cell-mediated immune (CMI) response, which normally eradicates infected cells by direct lysis. To maintain a chronic infection HIV must evade lysis by both cytotoxic T lymphocytes (CTLs), and natural killer (NK) cells. CTLs recognize infected cells with receptors that detect foreign peptide antigens presented in association with host major histocompatibility class I protein (MHC-I). NK cells recognize cells with abnormally low MHC-I levels and/or those that have upregulated NK activating ligands. Our goals are to better understand viral mechanisms of immune evasion and to ultimately inhibit these processes.
To evade the CTL response, HIV encodes the Nef protein, which inhibits CTL recognition by binding MHC-I molecules and recruiting cellular trafficking proteins, which prevent normal expression on the cell surface. In addition, we have found that CTL recognition is determined by how much viral protein is expressed by infected primary T lymphocytes. We have demonstrated that this is determined primarily by the relative activity of HIV Rev, which dials up the amount of HIV protein to the level that sustains viral production, while still allowing CTL immune evasion. These findings will aid in the development of Nef inhibitors, which will augment existing therapies by allowing anti-HIV CTLs to more efficiently recognize and lyse infected cells. In addition, insights into the cellular processes targeted by HIV will identify key elements of the immune system most important for control and eradication of viral infections.
As described above, the HIV Nef protein allows infected cells to evade recognition and lysis by reducing cell surface expression of MHC-I, which normally functions to present antigens to CTLs. In Nef-expressing cells we have shown that Nef binds to the cytoplasmic tail of newly synthesized MHC-I early in the endoplasmic reticulum as new antigens are being loaded. The Nef-MHC-I complex is transported normally to the trans-Golgi network (TGN), but is then diverted into the endolysosomal pathway, instead of going to the cell surface. Eventually, the MHC-I is targeted for destruction in lysososmal compartments. Our work has demonstrated that AP-1 expression is necessary for this pathway and indeed that Nef promotes the association of AP-1 with the MHC-I cytoplasmic tail in HIV-infected primary T lymphocytes.
Formation of the Nef-MHC-I-AP-1 complex results in the re-direction of MHC-I trafficking in such a way that it is targeted to lysosomes for degradation. However, cellular proteins that normally bind AP-1 are transported to endosomal compartments and recycled to the TGN. Thus, we hypothesized that another cellular factor may be required for the final step of endosome-to-lysosome transport. We have found that Nef interacts with another trafficking factor, b-COP in T lymphocytes, that it is essential for degradation of other cellular factors and that b-COP is necessary for targeting MHC-I to lysosomal compartments. Thus, Nef disrupts the trafficking of MHC-I by acting as an adaptor protein that links MHC-I to cellular trafficking proteins that promote its degradation.
As a result of the effects of Nef on antigen presentation, HIV-infected cells have low MHC-I cell surface expression, which could result in increased sensitivity to killing by NK cells. This is because NK cells become activated when their inhibitory receptors fail to detect sufficient MHC-I on the surface of target cells. Selective effects of the Nef protein on certain MHC-I molecules may help reduce susceptibility to NK cell. However, it has recently been discovered that NK cell lysis also requires stimulation through activating receptors, which bind ligands upregulated by a number of stresses such as DNA damage and viral infection. One such activating receptor, NKG2D, recognizes a host of structurally similar but distinct stress-inducible ligands. The human NKG2D ligands that have been identified to date include UL-16 binding proteins 1-4 (ULBP1-4) and MHC-I-related proteins A and B. There is some evidence that HIV infection can upregulate a subset of these ligands late in infection. We are currently exploring the viral mechanisms involved in limiting NK recognition and lysis.
Finally, to avoid immune recognition, a small proportion of HIV-infected cells achieve true viral latency, in which no viral proteins are expressed that could serve as antigens. Under certain conditions, gene expression can become activated in these cells and infectious virus can be made. The existence of such cells may help explain the observation that people with “undetectable” viral loads on anti-HIV therapy still have “blips” of virus production that are identifiable by certain ultra-sensitive techniques. In a substantial proportion of these people, this virus is clonal in nature, indicating that it came from the same infection and integration event, rather than by viral propagation and spread . It is crucial to identify populations such as this and to develop techniques to eradicate them in order to cure HIV disease.
We have found that freshly isolated CD34+ cells purified from human bone marrow take up and integrate viral genomes when exposed to HIV in vitro. However, after about three days in culture, only a small percentage of the cells expressed HIV gene products. The subsequent addition of factors that induced maturation along the myeloid lineage (PMA or GMCSF and TNF-alpha) resulted in a marked increase in the number of cells expressing HIV gene products and producing viral particles. Thus we have acquired substantial evidence supporting a role for progenitors as a long-lived inducible reservoir of HIV that is resistant to eradication by the immune system.
Our future goals will be to develop and define the key mechanisms outlined above through which HIV establishes a persistent infection. In this manner, we will uncover basic understanding of the innate and adaptive arms of the immune system while we forge inroads towards the development of novel antiviral therapeutics